First published online 30 June 2004
doi: 10.1242/dev.01239
Development 131, 3627-3636 (2004)
Published by The Company of Biologists 2004
Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination
Jennifer Schmahl1,2,
Yuna Kim1,
Jennifer S. Colvin3,
David M. Ornitz3 and
Blanche Capel1,*
1 Department of Cell Biology, Duke University, Durham, NC 27710, USA
2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA
3 Department of Molecular Biology and Pharmacology, Washington University
Medical School, St Louis, MO 63110, USA
View larger version (104K):
[in a new window]
|
Fig. 1. Fgf9-/- XY gonads on the C57BL/6 background do not form
testis cords. Testis cords are visible in wild-type XY gonads by 12.5 dpc
(A,C) and consist of groups of germ cells (red, detected with an antibody
against PECAM) surrounded by a layer of Sertoli cells and enclosed by a basal
lamina (green, detected with an antibody against laminin). PECAM (red) also
labels cells in the vasculature. Fgf9-/- XY gonads on the
C57BL/6 background did not form testis cords (B,D). In wild-type XY gonads,
SF1 (green) is detected in the nuclei of Leydig precursors (ley) outside
testis cords and, at a lower level, in the nuclei of Sertoli cells (s) within
testis cords at 12.5 dpc (examples indicated in E). In
Fgf9-/- XY gonads, a smaller number of SF1-positive cells
were detected, located near the surface of the gonad (F). Laminin (green) and
germ cell (red) localization in Fgf9-/- XX gonads at 12.5
dpc (H) were indistinguishable from Fgf9+/- XX gonads (G).
Scale bars: 50 µm.
|
|
View larger version (129K):
[in a new window]
|
Fig. 2. Proliferation is reduced in early Fgf9-/- XY gonads.
(A-C) Proliferating cells (red) were concentrated at and near the surface of
the XY gonads, examples indicated by arrows. At 11.2 dpc, most somatic cell
proliferation was observed in cells expressing SF1 (green). By 11.5 dpc, the
wild-type XY gonad underwent an increase in the amount of proliferation
observed at and beneath the surface of the gonad. (D-F) Fewer proliferating
cells were observed in Fgf9-/-XY gonads at all stages
examined. (G-I) In wild-type XX gonads, most proliferation was observed in
SF1-expressing cells at and near the surface of the gonad. No increase in
proliferation is observed at these stages. (J-L) Proliferation in
Fgf9-/- XX gonads was not noticeably different from XX
gonads. Scale bar: 25 µm.
|
|
View larger version (42K):
[in a new window]
|
Fig. 3. Fgf9-/- XY gonads are smaller than
Fgf9+/- XY littermates by 12.5 dpc. (A) Using PECAM
labeling to define the size of the gonad, it was observed that the width of
Fgf9-/- XY gonads was less than
Fgf9+/- XY gonads and roughly the same as XX gonads at
12.5 dpc. (The width at the gonad's center is indicated by brackets.) (B) The
mean width of gonads from XY individuals (+/+ and +/-) was 277.4±12.5
µm (n=9). Gonads from Fgf9-/- XY littermates
were significantly smaller (Student's t-test P<0.01),
with a mean width of 202.9±14.2 µm (n=6). Gonads from
Fgf9-/- XX individuals had a mean thickness of
187.3±6.8 µm (n=6) and were not significantly smaller than
wild-type XX gonads (193.5±8.5 µm, n=8,
P>0.05). Error bars indicate s.e.m. (C) The mean lengths of gonads
from XY (+/+ and +/-) and Fgf9-/- XY individuals were not
significantly different from each other (1016.3±28.4 µm and
981.5±30.2 µm, respectively, P>0.05). Additionally, the
mean lengths of gonads from XX (+/+ and +/-) and Fgf9-/-
XX individuals were not significantly different from each other
(997.5±13.8 µm and 1025.0±12.7 µm, respectively,
P>0.05).
|
|
View larger version (30K):
[in a new window]
|
Fig. 4. Fgf9 expression. (A) Using in situ hybridization, Fgf9
was expressed in both XY and XX gonads (g) during early stages of gonad
development (11.5 dpc). By 12.5 dpc, Fgf9 was specific to testis
cords in XY gonads and not detected in XX gonads. In the neighboring
mesonephros (m), Fgf9 was expressed within the mesonephric duct and
tubules (mt, arrows) of both sexes. (B) RT-PCR showed that FGF9 transcript was
present in both XX and XY gonads at 11.5 dpc, but was specific to XY gonads by
12.5 dpc. Reactions without reverse transcriptase served as a control.
Hprt and Fgf9 were amplified together in each tube, with
expected sizes of 325 bp and 400 bp, respectively.
|
|
View larger version (101K):
[in a new window]
|
Fig. 5. Location of FGF receptors. The FGF receptors 1, 3 and 4 (green) were found
in many somatic cells, and, at a higher level, in germ cells (red) in both XX
and XY gonads at 11.5 dpc. FGFR1 (A,B) and FGFR4 (E,F) were concentrated at
the cell surface, while FGFR3 (C,D) was found within the cell. FGFR2 (G,H) was
detected in somatic cells at and near the coelomic epithelium in both XY and
XX gonads (arrows). FGFR2 was also detected in the nuclei of scattered cells
within the XY gonad (G, arrowheads). Nuclear FGFR2 was not observed in XX
gonads (H). The number of cells with nuclear FGFR2 increased in the interior
of the XY gonad between 11.0 and 11.6 dpc (arrows in I-K). Scale bar: 50
µm.
|
|
View larger version (106K):
[in a new window]
|
Fig. 8. FGF9 does not induce Sertoli markers but does induce Scc. Nuclear
FGFR2 (green) was specific to Sertoli cells within the cords of wild-type XY
gonads at 12.5 dpc (A). In Fgf9-/- XY gonads, FGFR2 was
found in somatic cells throughout the gonad, but was not found in the nuclei
of any cells (C), in a similar pattern to the Fgf9+/- or
Fgf9-/- XX gonad (B,D). (E-G) FGFR2 is nuclear in cultured
XY gonads (E), but not in XX gonads (F). FGF9 did not induce the nuclear
localization of FGFR2 in cultured XX gonads (G). Germ cells and vasculature
are red. (H) Using in situ hybridization, Scc labels Leydig cells in
the interstitium of XY gonads and Sox9, Amh (Mis in figure)
and Dhh label Sertoli cells within testis cords. These genes were not
expressed in XX gonads at this stage. FGF9 did not induce markers associated
with Sertoli cell differentiation, but did induce Scc expression in
XX gonads. Scale bars: 50 µm in A-G; 250 µm in H.
|
|
View larger version (97K):
[in a new window]
|
Fig. 6. FGF binding and induction of proliferation. Gonadal sections were treated
with exogenous human FGF9, which was detected with an antibody specific to the
human protein (green). Human FGF9 bound to cells near the coelomic epithelium
(arrowheads) in XY gonad sections (B), but not XX (A) or XY sections
pretreated with heparatinase (C). Using BrdU labeling, many dividing cells
(red) were observed within cultured XY gonads, concentrated in somatic cells
(labeled with WT1, green) at and near the coelomic surface (D). Fewer dividing
cells were observed in XX gonads (E). Addition of 50 ng/ml FGF9 in the culture
media induced greater numbers of dividing cells in XX gonads. These dividing
cells were concentrated at the surface of the gonad (arrows). Germ cells and
vasculature are blue.
|
|
View larger version (97K):
[in a new window]
|
Fig. 7. FGFR2 colocalizes with SRYEGFP and SOX9 in Sertoli cell
precursors. In XY gonads at 11.5 dpc, nuclear FGFR2 (green) was detected in
cells expressing EGFP under the control of the Sry promoter (red,
found within the nucleus and cytoplasm). Cells where FGFR2 and EGFP colocalize
are indicated by arrows (A). The EGFP reporter is also expressed in XX gonads.
FGFR2 is detected in XX gonads at the coelomic surface, but is not found
within the nucleus (B). Nuclear FGFR2 (green) was found in every nucleus
labeled with SOX9 (red; C), while neither nuclear FGFR2 nor SOX9 was detected
in the XX gonad (D). Blood cell autofluorescence was observed in both blue and
red channels, and is pink. Red and green channels are separated in E-G to show
the overlap of FGFR2 (green) and SOX9 (red). Germ cells and vasculature are
labeled with PECAM and are blue. E-G are 200x magnification of the gonad
shown in C. Scale bar: 25 µm.
|
|
© The Company of Biologists Ltd 2004
